Movement of Molecules through Membranes

Page 196
Evidence is accumulating that the fluidity of cellular membranes can change in response to changes in diet or physiological state. Fatty acid and cholesterol content of membranes is modified by a variety of factors. In addition, pharmacological agents may have a direct effect on membrane fluidity. Anesthetics that induce sleep and muscular relaxation may have their action because of their effect on membrane fluidity of specific cells. A number of structurally unrelated compounds induce anesthesia, but their common feature is lipid solubility. Anesthetics increase membrane fluidity in vitro.
Thus cellular membranes are in a constantly changing state, with not only movement of proteins and lipids laterally on the membrane but with molecules moving into and out of the membrane. The membrane creates a number of microenvironments, from the hydrophobic portion of the core of the membrane to the interface with the surrounding environments. It is difficult to express in words or pictures the very fluid and dynamic state, in that neither captures the time­dependent changes that occur in the structure of biological membranes. Figure 5.27 attempts to illustrate the structural and movement aspects of cellular membranes.
Figure 5.27 Modified version of the fluid mosaic model of biological membranes to indicate the mobility of membrane proteins. t 0, t1, and t2 represent successive points in time. Some integral proteins (GP2) are free to diffuse laterally in the plane of the membrane directed by the cytoskeletal components, whereas others (GP ) may be 1
restricted in their mobility.
5.5— Movement of Molecules through Membranes
The lipid nature of biological membranes severely restricts the type of molecules that diffuse readily from one side to another. Inorganic ions or charged organic molecules do not diffuse at a significant rate because of their attraction to water molecules and exclusion of charged species by the hydrophobic environment of lipid membranes. The diffusion rate of carbohydrates, amino acids, and inorganic ions, however, is not zero but may be too slow to accommodate a cell's requirements for the substance. Where there is a need to move a substance across a cell membrane, specific mechanisms are available for its translocation.
The basic mechanisms by which molecules cross cellular membranes is presented in the following sections with examples of the processes for illustrative purposes. Specific systems are described in the context of individual metabolic processes in later chapters.
Some Molecules Can Diffuse through Membranes
Diffusion of a substance through a membrane involves three major steps: (1) solute must leave the aqueous environment on one side and enter the membrane; (2) solute must traverse the membrane; and (3) solute must leave the membrane to enter a new environment on the opposite side (Figure 5.28). Each step involves an equilibrium of solute between two states. Thermodynamic and kinetic constraints control the concentration equilibrium of a substance on two sides of a membrane and the rate at which it can attain equilibrium. Diffusion of gases such as O2, N2, CO2, and NO occur rapidly and depend entirely on the concentration gradient. Water diffuses readily through biological membranes; its movement occurs via gaps in the hydrophobic environment created by random movement of fatty acyl chains of lipids. Water and other small molecules move into these transitory spaces and equilibrate across the membrane from one gap to another. For diffusion of a solute with strong interaction with water molecules, the shell of water surrounding the solute must be stripped away before it enters the lipid milieu and then regained on leaving the membrane. Distribution of hydrophobic substances between the aqueous phase and lipid membrane will depend on the degree of lipid solubility of the substance; very lipid­soluble materials will dissolve in the membrane.
The rate of diffusion of a lipophilic substance is directly proportional to its lipid solubility and diffusion coefficient in lipids; the latter is a function of the size and shape of the substance. Uncharged lipophilic molecules, for exam­
Page 197
ple, fatty acids and steroids, diffuse relatively rapidly but water­soluble substances, for example, sugars and inorganic ions, diffuse very slowly.
Direction of movement of solutes by diffusion is always from a higher to a lower concentration and the rate is described by Fick's first law of diffusion:
where J is the net amount of substance moved per time, D is the diffusion coefficient, and d c/d x is the chemical gradient of substance. As the concentration of solute on one side of the membrane is increased, there will be an increasing initial rate of diffusion as illustrated in Figure 5.29. A net movement of molecules from one side to another will continue until the concentration in each is at chemical equilibrium. A continued exchange of solute molecules from one side to another occurs after equilibrium is attained but no net accumulation on one side can occur because this would recreate a concentration gradient if it occurred.
Figure 5.28 Diffusion of a solute molecule through a membrane. S1 and S2 are solutes on each side of the membrane, and Sm
is a solute in the membrane.
Movement of Molecules across Membranes Can be Facilitated
Mechanisms for membrane translocation of various substances including sugars, amino acids, metabolic intermediates, inorganic ions, and even H+ have been determined. The plasma membrane of both prokaryotic and eukaryotic cells, as well as membranes of subcellular organelles, contain transport systems that have an important role in the uptake of nutrients, maintenance of ion concentrations, and control of metabolism. These systems involve intrinsic membrane proteins and are classified on the basis of their mechanism of translocation of substrate across the membrane and the energetics of the system. A classification of transport systems is presented in Table 5.4. Each will be discussed in more detail in subsequent sections but for now it is important to distinguish the three main types.
Membrane Channels
Membranes of most cells contain specific channels, in some cases referred to as pores, which permit the rapid movement of specific molecules or ions from one side of a membrane to the other. The tertiary and quaternary structures of these intrinsic membrane proteins create an aqueous hole in the membrane that permits diffusion of substances in both directions through the membrane. Like diffusion, the substances will move only in the direction of lower concentration, that is, down a concentration gradient. In contrast to transporters, the channel proteins do not bind the molecules or ions to be transported. The
TABLE 5.4 Classification of Membrane Translocation Systems
Type
Class
Example
Channel
1. Voltage regulated
Na+ channel
2. Chemically regulated
Acetylcholine receptor
3. cAMP regulated
Cl– channel
4. Other
Pressure sensitive
Transporter
1. Passive mediated
Glucose transporter
2. Active mediated
a. Primary­redox coupled Respiratory chain linked
Primary­ATPases
Group translocation
b. Secondary
Na+, K+–ATPase
Na+­dependent glycose transport
Amino acid translocation
Figure 5.29 Kinetics of movement of a solute molecule through a membrane. The initial rate of diffusion is directly proportional to the concentration of the solute. In mediated transport, the rate will reach a Vmax
when the carrier is saturated.
Page 198
channels have some degree of specificity, however, based on the size and charge of the substance. Flow through the channel can be regulated by opening and shutting the passageway, like a gate to a garden.
Transporters
Transporters actually translocate the molecule or ion across the membrane by binding and physically moving the substance. The activity can be evaluated in the same kinetic terms as an enzyme­catalyzed reaction except no chemical reaction occurs. Transporters have specificity for the substance to be transported, frequently referred to as the substrate, have defined reaction kinetics, and can be inhibited by both competitive and noncompetitive inhibitors. Some transporters only move substrates down their concentration gradient (referred to as passive transport), while others can move the substrate against its concentration gradient (active transport) requiring the expenditure of some form of energy. With both channels and transporters the molecule is unchanged following translocation across the membrane.
A major difference between membrane channels and transporters is the rate of substrate translocation; for a channel, rates in the range of 107 ions s–1 are usual, whereas with a transporter the rate is in the range of 102–103 molecules s–1. The activity of all translocation systems can be modulated, permitting cells and tissues to control the movement of substances across membranes. Drugs for specific channels and transporters have been developed to control these processes.
Group Translocation
Group translocation involves not only movement of the substance across the membrane but also chemical modification of the substance during the process. One mechanism of uptake of sugars by bacteria involves transport and then phosphorylation of the sugar before release into the cytosol of the cell. In some mammalian cells uptake of amino acids involves a group translocation mechanism.
Membrane Transport Systems Have Common Characteristics
Membranes of all cells contain highly specific transporters for the movement of inorganic anions and cations (e.g., Na+, K+, Ca2+, HPO42+, Cl–, and HCO3–), and uncharged and charged organic compounds (e.g., amino acids and sugars). Different cellular membranes have different transport systems; as an example, the mitochondrial membrane has a specific mechanism to translocate ADP and ATP that is not present in other cellular membranes. Transport systems involve integral membrane proteins with a high degree of specificity for the substances transported. These proteins or protein complexes have been designated by a variety of names, including transporter, translocase, translocator, permease, and pump, or termed transporter system, translocation mechanism, and mediated transport system. The designations above are used interchangeably, but for convenience we will use transporter or translocase when referring to the proteins involved in translocation.
Membrane transporters have a number of characteristics in common. Each facilitates the movement of a molecule or molecules through the lipid bilayer at a rate that is significantly faster than can be accounted for by simple diffusion. If S1 is the solute on side 1 and S2 on side 2, then the transporter promotes establishment of an equilibrium as follows:
where the brackets represent the concentration of solute. If the transporter (T) is included in the equilibrium the rection is
Page 199
TABLE 5.5 Characteristics of Membrane Transporters
Passive Mediated
Active Mediated
1. Saturation kinetics
1. Saturation kinetics
2. Specificity for solute transported
2. Specificity for solute transported
3. Can be inhibited
3. Can be inhibited
4. Solute moves down concentration gradient
4. Solute can move against concentration gradient
5. No expenditure of energy
5. Requires coupled input of energy
If no energy is put into the system, the concentration on both sides of the membrane will be equal at equilibrium; but if there is an expenditure of energy, a concentration gradient can be established. Note the similarity of the role of a transporter to that of an enzyme; in both cases the protein increases the rate but does not determine the final equilibrium.
Table 5.5 lists major characteristics of membrane transport systems. As presented in Figure 5.29, they demonstrate saturation kinetics; as the concentration of the substance to be translocated increases, the initial rate of transport increases but reaches a maximum when the substance saturates the protein transporter. Simple diffusion does not have saturation kinetics. Constants such as Vmax and Km can be calculated for transporters. As with enzymes, transporters can catalyze movement of a solute in both directions across the membrane depending on the DG for the reaction.
Most transporters have a high degree of structural and stereo specificity for the substance transported. An example is mediated transport of D­glucose in erythrocytes, where the Km for D­galactose is 10 times larger and for L­glucose 1000 times larger than for D­glucose. The transporter has essentially no activity with D­fructose or disaccharides. Competitive and noncompetitive inhibitors have been found for many transporters. Structural analogs of the substrate inhibit competitively and reagents that react with specific groups on proteins are noncompetitive inhibitors.
There Are Four Common Steps in the Transport of Solute Molecules
We need to expand the equation above and consider four aspects of mediated transport (Figure 5.30). These are (1) recognition by transporter of appropriate solute from a variety of solutes in the aqueous environment, (2) translocation of solute across membrane, (3) release of solute by transporter, and (4) recovery of transporter to its original condition to accept another solute molecule.
The first step, recognition of a specific substrate by the transporter, is explained on the same basis as that described for recognition of a substrate by an enzyme. The presence of very specific binding sites on the protein permits the transporter to recognize the correct structure of the solute to be translocated.
The second step, translocation, is not completely understood. A reasonable mechanism (Figure 5.31) is one in which the protein transporter creates a channel between the environments on each side of the membrane with access through the channel being controlled by a gating mechanism in order to control which solutes can move into the channel. Transporters have receptor sites to which the solute attaches. After binding of solute and transporter, a conformational change of the protein moves the solute molecule a short distance, perhaps only 2 or 3 Å, but into the environment of the opposite side of the membrane. In this manner, it is not necessary for the transporter to move the molecule the entire distance across the membrane. Earlier suggestions for the translocation step included the possibility of a diffusible or rotating carrier, but both are improbable considering that transporters are large integral membrane proteins that do not diffuse transversely.
Figure 5.30 Reactions involved in mediated transport across a biological membrane. S1 and S2 are the solutes on sides 1 and 2 of the membrane, respectively; T and 1
T2 are the binding sites on the transporter on sides 1 and 2, respectively.
Figure 5.31 Model for a mediated transport system in a biological membrane. Model is based on the concept of specific sites for binding of substrate and a conformational change in the transporter to move the bound solute a short distance but into the environment of the other side of the membrane. Once moved, the solute is released from the transporter.
Page 200
Figure 5.32 Uniport, symport, and antiport mechanisms for translocation of substances. S and S 1represent different molecules.
Release, step 3, of the solute can occur readily if the concentration of solute is lower in the new compartment than on the initial side of binding. Without a change in the affinity (Keq), there would be a shift in the equilibrium and release of a portion of the solute. For those transporters that move a solute against a concentration gradient, release of the solute at the higher concentration requires a decrease in the affinity for the solute by the transporter. A change in the conformation of the transporter decreases the affinity. In group translocation (p. 210) the solute is chemically altered while attached to the transporter and the modified molecule has a lower affinity for the transporter.
Finally, in recovery, step 4, the transporter must return to its original state. If a conformational change has occurred, the transporter reverts to the original conformation.
The discussion above has centered on the movement of a single solute molecule by the transporter. There are systems that move two molecules simultaneously in one direction (symport mechanisms), two molecules in opposite directions (antiport mechanism), as well as a single molecule in one direction (uniport mechanism) (Figure 5.32). When a charged substance, such as K+, is translocated and no ion of the opposite charge is moved, a charge separation occurs across the membrane. This mechanism is termed electrogenic and leads to development of a membrane potential. If an oppositely charged ion is moved to balance the charge, the mechanism is called neutral or electrically silent.
Energetics of Membrane Transport Systems
The change in free energy when an uncharged molecule moves from a concentration of C1 to a concentration of C2 on the other side of a membrane is given by Eq. 5.1:
When DG is negative—that is, there is release of free energy—movement of solute will occur without the need for a driving force. When DG is positive, as would be the case if C2 is larger than C1, then there needs to be an input of energy to drive the transport. For a charged molecule (e.g., Na+) both the electrical potential and concentrations of solute are involved in calculating the change in free energy as indicated in Eq. 5.2:
where Z is the charge of the species moving, is the Faraday constant (23.062 kcal V–1 mol–1), and Y is the difference in electrical potential in volts across the membrane. The electrical component is the membrane potential and DG is the electrochemical potential.
A passive transport system is one in which DG is negative and the movement of solute occurs spontaneously. When DG is positive, coupled input of energy from some source is required for movement of the solute and the process is called active transport. Active transport is driven by either hydrolysis of ATP to ADP or utilization of an electrochemical gradient of Na+ or H+ across the membrane. In the first the chemical energy released on hydrolysis of a pyrophosphate bond drives the reaction, whereas in the latter an electrochemical gradient is dissipated to transport the solute.
Transport systems that can maintain very large concentration gradients are present in various membranes. An example is the plasma membrane transport system that maintains the Na+ and K+ gradients. One of the most striking examples of an active transport system is that present in the parietal cells of gastric glands, which are responsible for secretion of HCl into the lumen of the stomach (see p. 1069). The pH of plasma is about 7.4 (4 × 10–8 M H+), and